Recovery of hydrocortisone



States Patent RECOVERY OF HYDROCORTISONE Application November 26, 1954,Serial No. 471,514

4 Claims. (Cl. 260397.45)

No Drawing.

The present invention relates to a process for the separation of steroidmixtures particularly the mixtures resulting from biological oxidationof l7ix,21-dihydroxy-4-pregnene-3,20-dione and is more particularlyconcerned with the separation and recovery of 11/3,17-u,21-trihydroxy-4-pregnene-3,20-dione (hydrocortisone or Kendalls compound F) from amixture with l7a,21-dihydroxy-4-pregnene-3,20-dione (Reichsteinssubstance S) such as results from the oxidation of17a,21-dihydroxy-4-pregnene-3,20- dione by microorganisms.

The prior art discloses several processes, directed to the production ofl1/i,l7 z,21-trihydroxy 4 pregnene3,20- dione, which use as startingsubstance the readily available l7a,2l-dihydroxy-4-pregnene-3,20-dione.In these processes 17a,2l-dihydroxy-4-pregnene-3,20-dione (substance S)is brought into contact with microorganisms, illustratively CurvulariaInnate, U. S. Patent 2,658,023; Streptomyces fradiae, I. Am. Chem. Soc.74, 2381 (1952) or Cunninghamella blakesleeana U. S. Patent 2,602,769,and the like, which oxidize 17a,21-dihydroxy-4-pregnene- 3,20-dione toll B,l7o,2l-trihydroxy 4 pregnene-3,20- dione. Since these oxidationsare never complete, some of the starting material remains in thereaction mixture. Furthermore other oxygenated derivatives of thestarting material may occur, illustratively cortisone (compound E) andmaterials more or less polar than hydrocortisone and cortisone which areproduced when substance S is contacted with organisms of the generaCunninghamella, Streptomyces or Curvularia. In the processes describedin Patents 2,658,023 and 2,602,769, the desired 11,8,17a,21-trihydroxy-4-pregnene-3,20-dione is separated from the reactionmixture by repeated extractions followed by chromatography in a columnpacked with a suitable solid material and development of the column.Such a procedure is cumbersome and expensive due to the large amounts oforganic solvents necessary andthe time involved in the development ofthe column and is thus generally unsuitable for, large scale industrialproduction. The present invention overcomes the above-described'ditficultie's by using a solvent selected from the group consisting ofethylene dichloride and mixtures of ethylene dichloride and methanolcontaining up to ten percent by volume of methanol to separate the11fi,17a,21-trihydroxy-4-pregnene-3,20-dione from 17a,2l-dihydroxy-4-pregnene-3,20-dione.

While chlorinated hydrocarbon alone or in the presence of other solventshave been suggested or have been used for the extraction of steroidsresulting from a microbiological oxidation, these solvents have neverbeen found suitable for the separation and recovery of 11,8,l7a,21-trihydroxy-4-pregnene-3,20-dione without necessitating the additionaluse of a chromatographic column.

In the instant invention however the 113,l7a,2l-trihydroxy-4-pregnenel3,20-dione is separated from the solidmixture containing 11 9,17a,21-trihydroxy 4 pregnene- 3,20-dione,17a,21-dihydroxy-4-pregnene-3,20-dione, various other steroidsillustratively cortisone and other oxygenation and reduction products ofS, and metabolic prod- Patented June 4, 1957 ucts by extracting thismixture with a solvent selected from the group consisting of ethylenedichloride and mixtures of ethylene dichloride and methanol containingup to ten percent by volume of methanol, followed by cooling andfiltration of the precipitated11fl,17a,2l-trihydroxy-4-pregnene-3,20-dione.

It is an object of the present invention to provide a simplified,economic method to separate and recoverl1,8,17a,21-trihydroxy-4-pregnene-3,20-dione from a mixture containing17u,21-dihydroxy-4-pregnene-3,20-dione. It is particularly an object ofthe present invention to provide a method for the separation of11/3,17u,21-trihydroxy-4-pregnene-3,ZO-dione from a mixture obtainedfrom the oxidation of 17u,21-dihydroxy-4-pregnene-3,20- dione bymicroorganisms, wherein the use of chromatographic columns is avoided.Other objects of the present invention will be apparent to those skilledin the art to which this invention pertains.

The starting materials of the present invention are crude solid mixturesconsisting of hydrocortisone, 17oc,21-Clihydroxy-4-pregnene-3,20-dioneand other oxidation and reduction products of17o,2l-dihydroxy-4-pregnene-3,20- dione, illustratively, cortisonetogetherv with material of greater or lesser polarity than either17a,21-dihydroxy-4- pregnene-3,20-dione or11,8,1711,2l-trihydroxy-4-pregnene- 3,20-dione (hydrocortisone) andmetabolic products produced by the selected species of microorganisms.The amount of hydrocortisone in the mixture commonly varies betweenabout twenty to about percent and is usually between about forty andabout sixty percent. The amount of residuall7u,2l-dihydroxy-4-pregnene-3,20-dione is usually between about zero toabout twenty percent and most commonly is found to be between about fiveand about ten percent. The thus-described mixture may be obtained byvarious methods, illustratively by filtering the reaction mixtureresulting from fungus biooxidation to remove the mycelium and suspendedsolids, extracting the filtrate with organic solvents, preferably by acountercurrent Podbielniak extractor with ethyl acetate, concentratingthe thus obtained ethyl acetate solution in vacuo at low temperatures toobtain a cake, and extracting the cake with a hydrocarbon solvent suchas petroleum ether, Skellysolve B (a mixture of hexanes), to remove alarge proportion of fats.

In carrying out the process of the present invention the mixturecontaining 11B,17u,21-trihydroxy 4 pregnene- 3,20-dione,l70:,21-dihydroxy-4-pregnene-3,20-dione and other steroids produced byoxidation or reduction of l7a,2l-dihydroxy 4- pregnene-3,20-dione,illustratively cortisone, as well as some remaining fat and fatty acidderivatives is dissolved in a solvent selected-from the group consistingof ethylene dichloride and mixtures of ethylene dichloride with up toten volume percent of methanol. The ratio of solvent to solid mixtureused is based on the content of 11,6,17a,21-trihydroxy-4-pregnene-3,20-clione in the mixture as determined by quantitative chromatographyof a small sample of the crude mixture. Thus, if ethylene dichloridealone is employed, about one liter of solvent is used for each fourteengrams of 11,6,17a,2l-trihydroxy-4-pregnene-3,ZO-dione in the mixture,while if ethylene dichloride with methanol is used, about one liter ofsolvent is used for each forty grams ofl1,8,17a,21-trihydroxy-4-pregnene-3,ZO-dione in the mixture. In anyevent the total amount of solvent is suitably large enough to produceessentially total dissolution of the mixture at an elevated temperature,for example, at the boiling point of the solution. The thusobtainedsolution is then filtered and subsequently cooled. Cooling may beproduced by refrigeration, cold water and/or ice. If the cold water isinadequate to cool the solution more cooling can be achieved byvolatilizing some of the solvent through application of a vacuum. Aconvenient temperature range for crystallization is from minus twenty toplus twenty degrees centigrade, preferably between minus fifteen to plusten degrees centigrade. In order to facilitate crystallization ofhydrocortisone, mechanical stirring may be utilized. However,crystallization proceeds without stirring. After the crystallization isterminated, the solution containing the precipitated hydrocortisone isfiltered, and the crystals are dried to furnish 92 to 98 percent purehydrocortisone.

The following examples are illustrative of the process and products ofthe present invention, but are not to be construed as limiting.

Example 1 33.36 grams of a mixture containing 1047 grams ofhydrocortisone, 3.83 grams of cortisone and 1.38 grams of1711,21-dihydroxy-4-pregnene-3,20dione were admixed with 700 millilitersof ethylene dichloride and refluxed for ten minutes. The thus obtainedsolution was filtered while hot and then chilled to five degreescentigrade. The resulting crystals were separated by filtration anddried to yield 7.007 grams of hydrocortisone which was 93.2 percentpure.

Example 2 713.6 grams of a mixture containing 344.7 grams ofhydrocortisone and 149.8 grams of cortisone were dissolved in 26 litersof ethylene dichloride at reflux temperature. After the material hadessentially passed into solution, the solution was filtered with Celitediatomaceous earth filter-aid and the thus obtained clear filtrate wascooled with cold water and stirred for eighteen hours. To further coolthe solution to about ten degrees centigrade one liter of ethylenedichloride was removed by placing the solution under a vacuum andsupplying no heat. The thus obtained crystals were removed and dried atforty to fifty degrees centigrade at 28 inches of vacuum to yield 309grams of material which was shown to be 92.4 percent hydrocortisone asdetermined by quantitative paper chromatography. The following physicalconstants were found: Melting point 217 to 218 degrees centigrade;specific rotation [:1 of plus 169 degrees; E242 of 15,750; ash of 0.02percent and moisture of 0.5 percent.

Example 3 Four grams of a mixture containing 71 percent hydrocortisoneand seven percent cortisone were dissolved in 75 milliliters of ethylenedichloride and seven milliliters of methanol, the mixture was heated,filtered and chilled. 1.9 grams of hydrocortisone was obtained from thesubsequent filtration. This product contained 96.8 percent ofhydrocortisone and melted at 217 to 218 degrees centigrade.

Example 4 Nine thousand six hundred and seventy-four (9,674) grams of acrude mixture containing, as determined by paper chromatography, 4.982grams of hydrocortisone, 1548 grams of cortisone, and 774 grams ofl7a,2l-dihydroxy-4-pregnene-3,20-dione were dissolved in a mixtureconsisting of 130 liters of ethylene dichloride and twelve liters ofmethanol. The mixture was heated, filtered, and chilled to minusthirteen degrees centigrade. The thus obtained precipitate was removedby filtration and consisted of 2,842 grams of hydrocortisone which was98.1 percent pure.

It is to be understood that the invention is not to be limited to theexact details shown and described as obvious modifications will beapparent to one skilled in the art, and the invention is therefore to belimited only by the scope of the appended claims.

We claim:

1. A process for the separation and recovery of11B,170:,21-trihydroxy-4-pregnene-3,20-dione in a mixture with 171,2l-dihydroxy-4-pregnene-3,20-dione which comprises: dissolving saidmixture in hot solvent selected from the group consisting ofdichloroethylene and dichloroethylene with up to ten volume percent ofmethanol, cooling the solution to a temperature between about minustwenty and about plus twenty degrees centigrade and separating the thusprecipitated ll,B,l7a,2l-dihydroxy-4- pregnene-3,20-dione.

2. A process for the separation and recovery of11B,17a,21-trihydroxy-4-pregnene-3,ZO-dione from a mixture resultingfrom the biooxidation of l7a,21-dihydroxy- 4-pregnene-3,20-dione whichcomprises: dissolving said mixture in a solvent selected from the groupconsisting of dichloroethylene and dichloroethylene with up to tenvolume precent of methanol at about the boiling temperature of thesolution, cooling the solution to a temperature between about minusfifteen and about plus ten degrees centigrade and collecting the thusprecipitated l119,17a,2l-dihydroxy-4-pregnene-3,20-dione.

3. A process for the separation and recovery of11p,17a,21-trihydroxy-4-pregnene-3,20-dione from a mixture resultingfrom the oxidation of :,2l-dlhYdI'OXY-4- pregnene-3,20-dione bymicroorganisms which comprises: dissolving said mixture in hotdichloroethylene using about one liter of dichloroethylene for eachfourteen grams of 11,8,1701,21-trihydroxy-4-pregnene-3,20-dione in thesaid mixture, cooling thesolution to a temperature between about minusfifteen and about plus ten degrees centigrade and collecting the thusprecipitated 1lp,l7a,2l-trihydroxy- 4-pregnene-3,20-dione.

4. A process for the separation and recovery of1l5,17a,21-trihydroxy-4-pregnene-3,20-dione from a mixture resultingfrom the oxidation of l7a,2l-dihydroxy-4- pregnene-3,20-dione bymicroorganisms which comprises: dissolving said mixture in a hotsolution consisting of dichloroethylene with up to ten volume percent ofmethanol using one liter of solution for each forty grams of11,8,17a,21-trihydroxy-4-pregnene-3,20-dione contained in the saidmixture, cooling the solution to a temperature between about minusfifteen and about plus ten degrees centigrade and collecting the thusprecipitated 1lfi,17u,21- trihydroxy-4-pregnene-3,20-dione.

References Cited in the file of this patent UNITED STATES PATENTS2,602,769 Murray et al. July 8, 1952 2,658,023 Shull Nov. 3, 1953 OTHERREFERENCES MacArdle: Use of Solvents in Synthetic Org. Chem, pp. 13-14(1925).

1. A PROCESS FOR THE SEPARATION AND RECOVERY OF11B,17A,21-TRIHYDROXY-4-PREGNENE- 3,20-DIONE IN A MIXTURE WITH17A,21-DIHYDROXY-4-PREGNENE- 3,20-DIONE WHICH COMPRISES: DISSOLVING SAIDMIXTURE IN HOT SOLVENT SELECTED FROM THE GROUP CONSISTING OFDICHLOROETHYLENE AND DICHLOROETHYLENE WITH UP TO TEN VOLUME PERCENT OFMETHANOL, COOLING THE SOLUTION TO A TEMPERATURE BETWEEN ABOUT MINUSTWENTY AND ABOUT PLUS TWENTY DEGREES CENTIGRADE AND SEPARATING THE THUSPRECIPITATED 11B,17A,21-DIHYDROXY-4PREGNENE- 3,20-DIONE.